Characterization of an Evolutionarily Conserved Far-upstream Enhancer in the Human α2(I) Collagen (COL1A2) Gene

Abstract

We have examined the chromatin structure around and upstream of the transcriptional start site of the human ␣2(I) collagen (COL1A2) gene. Four strong DNase I-hypersensitive sites (HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. HS3, HS4, and HS5 were likewise mapped ϳ20 kilobases upstream of COL1A2 at about the same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1-12) and within stretches nearly identical to the mouse sequence. The region containing HS3-5 was found to confer high and tissue-specific expression in transgenic mice to the otherwise minimally active COL1A2 promoter. Characterization of the human element documented functional differences with the mouse counterpart. Enhancer activity substantially decreased without the segment containing FU1-7 and HS5, and inclusion of AluI repeats located 3 of HS3 augmented position-independent expression of the transgene. Hence, subtle differences may characterize the regulation of mammalian ␣2(I) collagen genes by evolutionarily conserved sequences.

Monocyte chemoattractant protein 3 as a mediator of fibrosis: Overexpression in systemic sclerosis and the type 1 tight‐skin mouse

“…There is substantial sequence conservation of both proximal and distal regulatory elements of the mouse and human pro␣2(I) collagen genes (23). We therefore confirmed our data on mouse constructs and also tested analogous human promoter-reporter constructs.…”

NFκB-Independent Signaling to the Cyclin D1 Gene by Rac

“…Therefore, there must be at least one essential Rac-regulated transcriptional element far upstream or downstream of the transcription start site. In fact, the collagen promoter is known to have distant enhancers located 20 kb upstream of the transcription start site, 45,46 while genes like Bmp2 47 and Sox9 48 have enhancer elements hundreds of kb downstream of their promoters. Although the identification of the critical Rac-regulated factor(s) requires further study, we can now suggest a model of cyclin D1 gene regulation in MEFs: ERK activity induces mid-G 1 cyclin D1 gene expression independently of NFκB activity while Rac and NFκB signal in cooperative, but parallel, pathways to stimulate cyclin D1 gene expression.…”

S-Adenosylmethionine Blocks Collagen I Production by Preventing Transforming Growth Factor-β Induction of the COL1A2 Promoter

“…AdoMet Prevents the Activation of the Mouse COL1A2 Promoter in Vivo-Transgenic mice harboring the Ϫ17 kb to ϩ54 bp of the proximal promoter of the mouse COL1A2 gene cloned upstream from the E. coli ␤-gal LacZ reporter gene have been shown to be valuable in studies of activation of the COL1A2 gene in vivo (14,16,18). CCl 4 activated the COL1A2 promoter as shown by the ␤-gal-positive blue staining ( Fig.…”